Receptor Complementation and Mutagenesis Reveal SR-BI as an Essential HCV Entry Factor and Functionally Imply Its Intra- and Extra-Cellular Domains

Abstract

HCV entry into cells is a multi-step and slow process. It is believed that the initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI), a major receptor of high-density lipoprotein (HDL), the CD81 tetraspanin, and the tight junction protein Claudin-1, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports have indicated that HDL promotes HCV entry through interaction with SR-BI. This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI. In contrast to CD81 and Claudin-1, the importance of SR-BI has only been addressed indirectly because of lack of cells in which functional complementation assays with mutant receptors could be performed. Here we identified for the first time two cell types that supported HCVpp and HCVcc entry upon ectopic SR-BI expression. Remarkably, the undetectable expression of SR-BI in rat hepatoma cells allowed unambiguous investigation of human SR-BI functions during HCV entry. By expressing different SR-BI mutants in either cell line, our results revealed features of SR-BI intracellular domains that influence HCV infectivity without affecting receptor binding and stimulation of HCV entry induced by HDL/SR-BI interaction. Conversely, we identified positions of SR-BI ectodomain that, by altering HCV binding, inhibit entry. Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement. Altogether, we demonstrate that SR-BI is an essential HCV entry factor. Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection.

Functional and Biochemical Characterization of Hepatitis C Virus (HCV) Particles Produced in a Humanized Liver Mouse Model

“…, mouse SR-BI (mSR-BI), or the hSR-BI point mutant G420H/ G424H were generated and expressed in Huh-7.5 cells as described previously (24). Down-regulation of hSR-BI was achieved using two small interfering RNAs (5Ј-GCAGCAGG-UCCUUAAGAAC and 5Ј-GGCACTGTTCTGGAACCTTC) as described previously (38).…”

“…The main physiological function of SR-BI is to transfer lipids between lipoproteins and cells (48), a function that is pivotal for HCVcc cell entry (23,24,47). We thus investigated the impact of the overexpression of the G420H/G424H hSR-BI mutant, which has impaired capacity to transfer lipids despite normal HDL binding capacity (49), on infectivity of HCVfrg and HCVcc subpopulations.…”

“…Moreover, other proteins implicated in cholesterol uptake such as Niemann Pick C-1-like 1 (21), and host cell kinases such as the epidermal growth factor receptor and the ephrin receptor A2 (22) have been identified as cofactors of HCV entry. Using the HCVcc and HCV pseudoparticle models, our laboratory recently demonstrated that SR-BI mediates entry of particles independently of their density via its capacity to transfer lipids to and from different carriers (23,24). Finally, presumably at a further step of entry, HCVcc particles of lower-intermediate density (i.e.…”

“…Finally, presumably at a further step of entry, HCVcc particles of lower-intermediate density (i.e. 1.08 -1.13 g/ml) and HCV pseudoparticles become able to bind SR-BI via a direct interaction with the E2 glycoprotein, which results in enhancement of their entry process (23)(24)(25).…”

Entry and replication of recombinant hepatitis C viruses in cell culture

“…Importantly, the tools are now available to study individually the 4 main HCV receptors (CD81, SR-BI, Cldn1 and Ocln): cell lines expressing very low levels of one of the receptors and retroviral vectors to rescue the expression of this receptor or one of its homologs have been developed in several laboratories [44,101,102,104,106,116,165] (see Table 1). Thanks to HCVpp, it is also possible to extend these receptor-complementation studies in cell lines that poorly replicate HCV, since the readout of HCVpp infectivity is independent on HCV replication [113,116,119].…”